The current invention pertains to biarylamino compounds which are useful as highly selective inhibitors of certain protein tyrosine kinases, PTKs, which are one of the sub-classes of the protein kinases, PKs. PKs are very important signaling entities in intracellular communication, where they modify many proteins by catalyzing the transfer of a phosphate group from ATP acting as a phosphodonor to a phenolic hydroxyl on a tyrosine side chain of the protein. Frequently, the tyrosine kinases are incorporated into the intracellular domain of a very large transmembrane protein, which has a cognate ligand binding domain in the extracellular domain, whereby ligand binding activates the tyrosine kinase intracellularly. Such molecules are receptor tyrosine kinases (RTKs).
Structurally, the kinases are quite well understood. There is a kinase domain, which may be the whole protein, or only one domain of a much larger modular protein, and this domain has a basic conserved structure of about 35 kD, consisting of two lobes, the N-terminal one being mainly made up of β-sheets, and the larger C-terminal domain mainly of α-helices. There is a deep cleft between the two lobes which binds both ATP and the substrate. The substrate binding domain is quite large, and rather variable, and is used to discriminate between different protein substrates, and maintain specificity of phosphorylation. This specificity can be very variable, with some enzymes such as MEK having only one known substrate, and others being able to phosphorylate hundreds of distinct hydroxyls in proteins.
Phosphorylation frequently changes the conformation of the modified protein, often converting enzymes from an inactive form to an active form, or vice versa, or causing the protein to associate closely with specific binding partners, or perhaps dissociate from them, leading to changes in cellular localization, or assembly, or disassembly, of functioning multi-protein complexes. Many of the transducers of signals into cells, and from the cell surface into the nucleus are either PKs, or controlled by PKs, especially RTKs. Because of this, inhibitors of the kinase activity of PKs can have very drastic effects on cellular signaling, damping down both normal responses to external signals, and inappropriate overresponses, usually caused by mutations in the signaling molecules themselves. Although such pathways are very widespread in the body, and are involved in one way or another in most bodily functions, and the diseases that can arise from their malfunction, inhibitors of PKs are particularly useful in treating cancer and immunological disorders, both disease classes where overactivity of PKs, especially RTKs, has been widely documented, and where they often play crucial roles in driving the disease process itself.
Kinases have been shown to be very important effectors of many disease processes, especially in cancer. Cellular proliferation is controlled at many different levels by kinases, and, under normal circumstances for cells to proliferate, signals have to be sent from outside the cell, where they bind to receptors and activate the receptors. Many of the important receptors in cell signaling are kinases, especially RTKs, or are directly coupled to kinases which themselves are activated by the activated receptor. Once these kinases have been activated, they in turn activate signaling cascades, which usually involve several further kinases in an amplifying wave of phosphorylation, which lead eventually to the translocation into, and activation of, transcription factors in the nucleus. Activation of the transcription factors engenders proteins being produced which carry out various programs within the cell, including those which start the cell into the proliferative cycle. Usually, once this process has gone on for a number of hours, the newly synthesized proteins will continue the process, without need for further extracellular input. If the proliferative cell cycle is initiated, the first set of proteins synthesized includes both further transcription factors, and their activators to drive later stages of the cell cycle, and effectors, which start the process of duplicating and dividing the cell. Kinases are major controllers of every step in this process. When this process is not controlled properly, and cells can execute the cell cycle without appropriate external control, they become transformed, and can form a tumor, if the immune system fails to eradicate them.
When transformed cells are examined, one of their invariant characteristics is hyperphosphorylation, showing that these cells have an overall surfeit of kinase activity, especially in the absence of any growth factors. Hyperphosphorylation can be caused by a very wide variety of mutations in the cell. For example by the cell inappropriately producing its own ligand for one of the receptor-linked kinases. Or one of these kinases may be heavily overexpressed, due either to a failure to control its expression properly, or to multiple extra copies of the gene being present in the cell. Another very common genetic defect is a mutation in the coding region of the kinase, which leads to a kinase which is constitutively active, and has no need for the appropriate signal to active it. Sometimes the kinase may not be inappropriately active, but a phosphatase, which is supposed to limit its signaling by removing the phosphate from target molecules, is inactivated by mutation or deletion. Examination of both cell culture tumors and isolates from clinical tumors will almost always find defects of this sort in the phosphorylation system of the tumor cells.
In the late 1980s, several small molecule kinase inhibitors were discovered. These molecules almost invariably bind in the catalytic cleft of the kinase, and compete with ATP for its binding site. Thus they are ATP-competitive, and most inhibitors discovered since then fall into this class. However, kinase inhibitors have been occasionally discovered which compete with the protein substrate, substrate-competitive, or more commonly with both ATP and substrate, dual inhibitors, or are neither competitive with receptor nor substrate, non-competitive inhibitors. After allowing for differences in cellular penetration, one finds that there is a very good correlation between the potency of these compounds in isolated kinase enzyme inhibitory assays, and inhibition of the kinase in cells. For many kinases, there is also an excellent correlation between loss of phosphorylation of downstream targets, and inhibition of cellular proliferation. As this correlation has been shown thousands of times, with dozens of different kinases, it is a clear demonstration that aberrant kinase signaling can cause uncontrolled proliferation in transformed cells, and that in many cases, blockade of the over-activated kinase can stop the proliferation. In many cases the kinase inhibitor alone can actually induce apoptosis in the transformed cells, leading to shrinkage of the tumor. This can occur because various genetic lesions in the cell have been detected by the cellular proof-reading system, and as a result several pro-apoptotic mechanisms are usually activated in these cells, but aberrant phosphorylation may well be involved in suppressing the ongoing apoptotic process. Some kinase inhibitors, especially those which target kinases involved late in the cell cycle are intrinsically cytotoxic, as cells interrupted during mitosis tend to apoptose very readily. Although, good proof that these abilities in cells could prevent tumors grown as xenografts in nude mice was initially slow in coming, as the agents improved, it became routine to demonstrate that kinase inhibitors could slow the growth of tumors which express the kinase oncogenes being targeted, and the better agents cause the tumors to regress in size often to the point of immeasurability, and on rare occasions the tumors do not regrow after dosing is stopped, suggesting the animals may have been cured of the tumor. Furthermore, the in vivo efficacy correlates with the cellular and enzymatic activity, after one has correlated for tumor exposure.
Clinical proof was slower in coming, probably partly because clinical tumors are often much more complex than tumors grown under carefully controlled conditions, partly because mice are a lot more biochemically robust than humans, and can tolerate larger relative doses of the drugs, and mainly because it is usually very difficult to know which are the appropriate kinases to inhibit in any given randomly presenting human tumor. However imatinib, a reasonably potent inhibitor of the fusion oncogenic TK BCR-ABL, with truly outstanding pharmacokinetic properties, was approved for chronic myelogenous leukemia (CML) in 2000. This kinase inhibitor provides a very convincing clinical proof of concept for the theory, as about two thirds of CML patients (whose tumors almost by definition contain one of two forms of BCR-ABL) respond very well to treatment, and usually the leukemia cells almost completely disappear from circulation. Surprisingly, mutation around this blockade appears to be very slow, and even after 10 years of treatment the drug is still effective in 80% of patients. This has not proved to be the general case, probably partly because most tumors are found much later in their biological history than are CMLs, and have had much longer to become genetically heterogeneous, and partly because very few tumors are as dependent on one oncogene as CML is on BCR-ABL.
Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors
Two 4-anilinoquinazoline inhibitors of the epidermal growth factor RTK (EGFR, erbB-1), gefitinib and erlotinib, were approved for use in lung cancer around 10 years ago. EGFR is one of the most commonly disregulated kinases seen in solid tumors, with overexpression or mutation being seen often in 50% or more of a tumor type, including non-small cell lung cancer (NSCLC). Despite excellent activity of these inhibitors against a wide variety of xenografts overexpressing EGFR, very limited activity was seen in NSCLC, with only about 10% of patients responding to the drug, and the average response only lasting a year or so, although occasionally a much more durable responder is found. Surprisingly, in other tumor types known to overexpress EGFR, especially colorectal cancer (CRC) no meaningful activity was demonstrated, although the anti-EGFR monoclonal antibody Erbitux has shown quite good clinical activity in CRC, for which it has been approved for use.
When close examination of NSCLC responders was made, it was found that the majority of good responders had one of a few single mutations in EGFR (sm-EGFR), with those containing wild-type receptor (wt-EGFR) usually not responding appreciably, regardless of expression level. Such mutations are very rare in CRC, which tends towards overexpressed wt-EGFR, or overexpressed autocrine ligand expression. When these mutants, especially EGFR L858R, and EGFR de1746-750, were analyzed it was found that they have the properties of being both intrinsically activated, which means that they were driving proliferation without an external signal, and also binding ATP more weakly than wt EGFR, (higher Km) whilst having similar affinity to wt EGFR for the inhibitors. This meant that, as these inhibitors are ATP-competitive, that it was easier to compete ATP off the enzyme and shut down kinase activity in susceptible mutants than in the wt, giving a de facto boost to inhibitor potency in the mutants. At the same time these tumors had become more dependent on EGFR signaling for proliferation and survival than most tumors, because the signals had been reliably overactive ever since the original mutation event.
As stated earlier, solid tumors such as lung cancers are usually quite old by the time they are discovered, probably on average being 6-12 years beyond the arising of the original transformed founder cell. One of the properties of transformed cells is that they lose control over their DNA replication quality control, so their spontaneous mutation rate is much higher than that of untransformed cells. As mutations occur most easily during DNA replication, and these cells are replicating very quickly, this adds further to the mutation rate. The result is that as a tumor ages it will pick up an ever-increasing number of mutations, and it does so in a stochastic fashion, so that sub-clones of the tumor arise over time with somewhat different genetics from the original tumor, and one another. These sub-clones are not only involved in a survival struggle with the body itself, but with one another as they compete amongst themselves for the limited resources available to them. If one changes the environment for the dominant tumor clone, such that it becomes relatively less well adapted to its new environment, for example by adding an effective inhibitor to it, a previously much less successful minor clone may be able to take over the niche being vacated, if it is not as affected by said inhibitor. Alternatively, unless one either kills the clone outright, or completely shuts down proliferation, it will continue to spawn mutations, and if a mutation gets around the inhibition, this sub-clone will now be free to proliferate, without hindrance from either the inhibitor or the inhibited parental clone. Thus natural selection predicts that cancers, just like infectious diseases, should be able to develop drug resistance, and as the selection process is largely driven by competition between tumor sub-clones within a single host, the overall effect is to favor more aggressive sub-clones, and tumors generally become more deadly as they evolve.
When responders to gefitinib and erlotinib were followed, it was found that the onset of resistance could be correlated with several different genetic changes. In rare cases the tumors seem to pick up a totally different signaling system to drive the tumor, but usually the resistance involves tweaking of the original system. EGFR is a member of the erbB (Type I) subfamily of RTKs, along with erbB-2, erbB-3 and erbB-4. These receptors are activated by ligands which induce them to dimerize, and although EGFR-EGFR homodimers are quite commonly used in signaling, the more usual course in this family is for the ligands to induce heterodimerization, such that the signaling entity will be for example EGFR:erbB-2 or erb-B2:erbB-3 and an appropriate ligand. The simplest way to reactivate the system is to increase the expression of one of the other erbBs, and this is frequently seen, even before treatment, and may help to explain why a lot of wt EGFR overexpressing tumors do not respond to EGFR inhibition. A somewhat related mechanism involves the RTK HGFR, which although not a erbB family member has been shown to form oncogenic heterodimers with erbB family members, especially erbB-3, when overexpressed, and overexpression of HGFR is a common resistance mechanism to EGFR inhibitors. At least in laboratory settings, addition of an HGFR inhibitor to these cells restores sensitivity to EGFR inhibitors. The third, and commonest, mode of resistance is a further mutation in EGFR, giving doubly mutant receptor (dm-EGFR) which reduces its sensitivity to the EGFR inhibitor. The commonest of these is the so-called “gatekeeper” mutation T790M, and NSCLCs with double mutants such as L858R/T790M are commonly seen in initial responders, who have subsequently developed resistance to EGFR inhibitors. Whether such sub-clones were present all along, or whether they only arise after treatment is not known, but it seems most probable that the mutation is already present in short term responders, and may arise as a de novo mutation in long term responders who develop resistance late.
Initially, it was believed that these mutations block the inhibitors sterically from binding to the mutant enzyme, hence reducing their affinity, and efficacy. However, more recent studies suggest that the commonest mutations have very little effect on inhibitor affinity, but lead to restoration of ATP-binding affinity to that of wt EGFR, or possibly up to 10-fold greater, with the result that the achievable concentrations of the inhibitors are no longer high enough to shut down signaling to a therapeutically useful extent. In principle, one simply needs to improve the affinity of the inhibitors enough to overcome the increased ATP affinity, but in practice this is very difficult to do, because gefitinib and erlotinib are already very potent, subnanomolar, EGFR inhibitors with good PK properties, and yet have mediocre activity against tumors driven by wt EGFR. Furthermore, although the T790M mutant does not reduce the affinity of EGFR for erlotinib and gefitinib, it does limit the ways that one could increase affinity in the anilinoquinazoline chemotype of these two inhibitors. Therefore, to find greater affinity for the T790M-type mutants, new chemical templates have been examined, and some, especially U-shaped inhibitors of the type discussed later, appear to have considerable promise in this area.
EGFR receptors play an important role throughout the body, especially in the entire gastrointestinal epithelium and skin, which are both proliferatively very active tissues. As two of the major, dose-limiting toxicities of EGFR inhibitors are skin rashes and serious GI disturbances, these are almost certainly largely mechanism-based toxicities. As long as the tumor is driven by wt EGFR this is very difficult to avoid by rational design, especially for an oral agent, where GI tract exposure is obligate, but if the tumor is driven by mutant EGFR, one may be able to mitigate the toxicity seen with the approved drugs. For NSCLCs which respond to EGFR inhibitors, the initial target is not wt-EGFR, but one of a limited number of sm-EGFRs, and the later target is a dm-EGFR, both of which should at least in principle have different SARs to wt-EGFR, giving one at least the theoretical possibility of reducing side effects by finding inhibitors which have considerably better affinity for sm- and/or dm-EGFR over wt-EGFR. Due to the similarity between EGFR and the mutant-EGFRs, and the fact that the original inhibitors only worked because they already were better inhibitors of sm-EGFR than wt-EGFR, not due to intrinsic affinity, but ATP-competition, this might be expected to be a difficult feat to accomplish. Unfortunately, clinical observation suggests that the aberrant EGFR systems driving tumors need to be very heavily suppressed to produce meaningful efficacy, whereas the suppression of wt-EGFR signaling in normal tissues at high enough levels to induce limiting toxicities is relatively easy to accomplish. However EGFR inhibitors with enhanced affinity for EGFR mutants, especially T790M dm-EGFRs have been found and examples of many of these are in the literature, with several now in clinical trials. This patent application describes compounds which fit one of these criteria.
Inhibitors of EGFR which have considerably greater affinity for a mutant EGFR than the wt EGFR should at an optimal dose be able to inhibit proliferation in tumors driven by that mutant, whilst having relatively little, if any effect on EGFR signaling in untransformed tissues, where wt EGFR is responsible for the EGFR signaling. This should allow considerably larger doses of mutant-selective EGFR inhibitors to be given, increasing both the efficacy against the mutant-driven tumor and the therapeutic index. It should be noted that because of mutant effects on ATP-binding, that is essentially what is already happening with responders to erlotinib and gefitinib, where the responding mutants are actually more sensitive to the inhibitors than wt EGFR, due mainly to their diminished affinity for the competing ligand ATP. Several third generation EGFR inhibitors have now been revealed, with some in the clinic. These compounds are generally irreversible inhibitors, initially based off of a U-shaped dianilinopyrimidine scaffold, but this been extended to several related scaffolds, but all bind in a similar mode to the dianilinopyrimidines. In general these compounds are very potent inhibitors of the mutant EGFRs, containing the T790M mutation, and are somewhat less potent against wt EGFR, and some of the other mutations. Because of this profile, it is believed that the mechanism-based toxicities of wt EGFR inhibition should be considerably reduced, while retaining very strong inhibitory potency against tumors driven by the appropriate EGFR mutations. Thus compounds of this type may be especially useful as second line therapy, after a patient previously sensitive to first line erlotinib or gefitinib therapy becomes resistant. Not only will these inhibitors allow the appropriate mutant receptors to be inhibited as strongly as previously, but they should do this whilst themselves not inducing appreciable mechanism-induced toxicity through EGFR inhibition. The macrocyclic amine inhibitors of the present invention are irreversible inhibitors of EFGR, with a similar selective profile for mutant over wt EGFR inhibition to these agents, and excellent pharmacokinetic properties, and will therefore prove to be excellent agents for second line treatment of NSCLC, and any other tumors driven by this sub-family of mutated EGFR kinases.
Another method of increasing the potency of especially EGFR inhibitors was developed in the mid-1990s. Many sites on proteins are quite strongly nucleophilic, either because they are intrinsically nucleophilic, with cysteine thiols being the principle example, with lysine amines, histidine imidazoles, and serine, threonine and tyrosine hydroxyls also being less potent possibilities, or because they have been deliberately activated, as in the catalytic hydroxyls in many amidases. Such residues can often be targeted by electrophiles, which modify the protein under rather mild conditions. Depending on the function of the modified residue, and its position on the protein, this may or may not lead to a loss of enzyme function. It was realized that a subset of TKs use a cysteine residue on the edge of the ATP binding cleft to form a hydrogen bond to the ribose of ATP, whereas the majority use a threonine for this purpose. The EGFR family all contains this cysteine (C797 in EGFR). It was hypothesized that this cysteine could be alkylated by an alkylating moiety attached to an inhibitor, which bound in the ATP-binding site, and presented the electrophile in the vicinity of the cysteine sulfur. Indeed many of the first generation of EGFR inhibitors were potent electrophiles, which may well have targeted Cys797 or other nucleophiles on EGFR. Unfortunately, this inhibition did not lead to very potent inhibitors, nor did it lead to very selective inhibitors, suggesting that the electrophiles were reactive enough, and non-discriminating enough to react with a wide variety of proteins, especially kinases, and that in many of these cases the alkylation was occurring in either the catalytic domain, or a controlling “switch region” of the enzyme. To make this concept useful, the alkylating moiety would have to be of low intrinsic reactivity, because one does not want it to indiscriminately react with the vast array of nucleophiles in the body, both for potential PK and toxicity reasons. To get an alkylating agent to react with this necessarily rather weak electrophile with high selectivity, it was shown that the compound itself had to have both high (non-covalent) affinity for the binding site, and would have to bind preferentially in a conformation which placed the weak electrophile in close proximity to the electrophile. Lastly, it was also found that the reaction needed to be fast relative to the plasma half-life of the inhibitor, or most of it would wash out of the body without ever reacting with the crucial cysteine. Such irreversibly inhibitory compounds were discovered, and it was found that they not only were much more potent inhibitors of EGFR in vivo than the theoretically equipotent reversible inhibitors, but as a bonus they made (at least in the case of the anilinoquinazolines and the related 3-cyanoquinolines) a rather poor erbB-2 and erbB-4 inhibitory template into very potent inhibitors of all of the erbBs, demonstrating that if the binding mode were really good in its placement of the alkylating moiety, very high non-covalent affinity for the target might be less vital. Most of the second generation EGFR inhibitors which went into the clinic are irreversible inhibitors of EGFR, using acrylamide derivatives as electrophiles, and they appear to be more active in general in the clinic than reversible inhibitors, but they also tend to have higher toxicity, so only one, afatinib, has shown a good enough profile to gain approval.
Many different classes of kinase inhibitors have been developed, and several have been successfully approved and marketed. One of the molecular scaffolds which appears to produce potent inhibition of a large number of kinases, is a series of three concatenated rings, of which two, and frequently all three, are aromatic, which can form a U-shaped structure when binding to a kinase. The two distal rings can be directly linked to the central ring by bonds, or via various linkers consisting of 1-3 atom chains. The central ring, which is almost invariably a nitrogen-containing heteroaromatic system with an NH group adjacent to a ring nitrogen, forms 1-3 hydrogen bonds to the backbone of residues in the hinge domain of kinases, between the N- and C-terminal lobes, just prior to the so called DFG loop, an invariant structure in kinases, which has to be placed correctly for an active conformation of the enzyme to be achieved. This end of the inhibitor also occupies a part of the adenine-binding region of the kinase, which tends to be very hydrophobic, whereas the two rings, which make the “stems” of the U, occupy a broad channel frequently filling part of the space normally occupied by the rest of the ATP molecule. Although quite a lot of affinity for specific kinases comes from decorating these core rings with selected substituents which produce favorable interactions with hopefully unique structural determinants in the target kinases, and/or unfavorable interactions with kinases, which one does not wish to inhibit, a lot of the affinity and selectivity for various kinases comes from the various torsions and bend angles between the three rings, and some substituents which optimize affinity for the target kinase may not themselves interacting directly with the protein, but may control the most stable conformations of the three rings with respect to one another. Thus the purpose of some substituents can be to affect the overall internal energy of the inhibitory molecule, in order to stabilize a favorable conformation for binding, rather than directly interact with the kinase.
None of the first and second generation EGFR/erbB-2 inhibitors which entered the clinic show the U-shaped binding mode. They have the 4-anilino (or extended 4-anilino) group binding into a cleft between the β4 sheet and the αC-helix, which is behind the vital L745-D855 salt bridge, and the DFG loop of which D855 is a part.